In this proposed research we are applying genetic and biochemical approaches to the study of the leucine transport in bacteria. We are determining the number of components in the high affinity and low affinity transport systems for leucine. The high affinity system requires a periplasmic binding-protein which we have isolated, purified, and crystallized. This component bears the receptor site for leucine. In collaborative studies the amino acid sequence of this component has been determined and currently the three-dimensional structure is being determined using x-ray crystallographic techniques. We are identifying additional components of the leucine transport system using genetic approaches. We determined that the low affinity membrane-bound transport system for leucine is a separate and distinct system operating in parallel with the binding-protein system. We are examining the regulation of leucine transport and the way this regulation influences the overall cell physiology. We are determining if the genes that concern the leucine transport form a transport operon and, if so, how it is regulated. An examination of the sensitivity of E. coli to addition of leucine to the growth medium and the possible role of transport in this sensitivity is underway. Finally, we are studying whether the leucine-binding protein is inserted into the periplasmic space in a random manner or whether only at specific times during the cell cycle. These studies are being carried out in parallel with similar studies on transport in animal cells grown in tissue culture so that techniques gained with bacteria can be applied to the study of transport in higher organisms. BIBLIOGRAPHIC REFERENCES: Quay, S. C., Dick, T. E., and Oxender, D. L. (1977) Role of Transport Systems in Amino Acid Metabolism: Leucine Toxicity and the Branched-Chain Amino Acid Transport Systems. J. Bacteriol. 129, 1257-1265. Anderson, J. J., and Oxender, D. L. (1977) Escherichia coli Transport Mutants Lacking Binding Protein and Other Components of Branched-Chain Amino Acid Transport Systems. J. Bacteriol. 130, in press.